Dual-Mechanism ERK1/2 Inhibitors Exploit a Distinct Binding Mode to Block Phosphorylation and Nuclear Accumulation of ERK1/2.

The RAS-regulated RAF-MEK1/2-ERK1/2 signaling pathway is frequently deregulated in cancer due to activating mutations of growth factor receptors, RAS or BRAF. Both RAF and MEK1/2 inhibitors are clinically approved and various ERK1/2 inhibitors (ERKi) are currently undergoing clinical trials. To date, ERKi display two distinct mechanisms of action (MoA): catalytic ERKi solely inhibit ERK1/2 catalytic activity, whereas dual mechanism ERKi additionally prevents the activating phosphorylation of ERK1/2 at its T-E-Y motif by MEK1/2. These differences may impart significant differences in biological activity because T-E-Y phosphorylation is the signal for nuclear entry of ERK1/2, allowing them to access many key transcription factor targets. Here, we characterized the MoA of five ERKi and examined their functional consequences in terms of ERK1/2 signaling, gene expression, and antiproliferative efficacy. We demonstrate that catalytic ERKi promote a striking nuclear accumulation of p-ERK1/2 in KRAS-mutant cell lines. In contrast, dual-mechanism ERKi exploits a distinct binding mode to block ERK1/2 phosphorylation by MEK1/2, exhibit superior potency, and prevent the nuclear accumulation of ERK1/2. Consequently, dual-mechanism ERKi exhibit more durable pathway inhibition and enhanced suppression of ERK1/2-dependent gene expression compared with catalytic ERKi, resulting in increased efficacy across BRAF- and RAS-mutant cell lines.

Fragment-Based Discovery of a Potent, Orally Bioavailable Inhibitor That Modulates the Phosphorylation and Catalytic Activity of ERK1/2.

Aberrant activation of the MAPK pathway drives cell proliferation in multiple cancers. Inhibitors of BRAF and MEK kinases are approved for the treatment of BRAF mutant melanoma, but resistance frequently emerges, often mediated by increased signaling through ERK1/2. Here, we describe the fragment-based generation of ERK1/2 inhibitors that block catalytic phosphorylation of downstream substrates such as RSK but also modulate phosphorylation of ERK1/2 by MEK without directly inhibiting MEK. X-ray crystallographic and biophysical fragment screening followed by structure-guided optimization and growth from the hinge into a pocket proximal to the C-α helix afforded highly potent ERK1/2 inhibitors with excellent kinome selectivity. In BRAF mutant cells, the lead compound suppresses pRSK and pERK levels and inhibits proliferation at low nanomolar concentrations. The lead exhibits tumor regression upon oral dosing in BRAF mutant xenograft models, providing a promising basis for further optimization toward clinical pERK1/2 modulating ERK1/2 inhibitors.

Defining and Measuring Patient Satisfaction.

Reporting patient satisfaction has become an increasingly common component of studies evaluating treatment outcomes. However the construct of "patient satisfaction" is one that is complex and context dependent. While there is no question that careful, reliable, and valid measurement of this important aspect of patient care is required, tools for achieving this objective have not been fully developed. Measures of patient satisfaction that reflect the unique role of the hand in everyday life will require the same approach to instrument development as has been used to move forward the field of outcome measurement in general.

Protease Inhibitors Block Multiple Functions of the NS3/4A Protease-Helicase during the Hepatitis C Virus Life Cycle.

UNLABELLED: Hepatitis C virus (HCV) NS3 is a multifunctional protein composed of a protease domain and a helicase domain linked by a flexible linker. Protease activity is required to generate viral nonstructural (NS) proteins involved in RNA replication. Helicase activity is required for RNA replication, and genetic evidence implicates the helicase domain in virus assembly. Binding of protease inhibitors (PIs) to the protease active site blocks NS3-dependent polyprotein processing but might impact other steps of the virus life cycle. Kinetic analyses of antiviral suppression of cell culture-infectious genotype 1a strain H77S.3 were performed using assays that measure different readouts of the viral life cycle. In addition to the active-site PI telaprevir, we examined an allosteric protease-helicase inhibitor (APHI) that binds a site in the interdomain interface. By measuring nucleotide incorporation into HCV genomes, we found that telaprevir inhibits RNA synthesis as early as 12 h at high but clinically relevant concentrations. Immunoblot analyses showed that NS5B abundance was not reduced until after 12 h, suggesting that telaprevir exerts a direct effect on RNA synthesis. In contrast, the APHI could partially inhibit RNA synthesis, suggesting that the allosteric site is not always available during RNA synthesis. The APHI and active-site PI were both able to block virus assembly soon (<12 h) after drug treatment, suggesting that they rapidly engage with and block a pool of NS3 involved in assembly. In conclusion, PIs and APHIs can block NS3 functions in RNA synthesis and virus assembly, in addition to inhibiting polyprotein processing. IMPORTANCE: The NS3/4A protease of hepatitis C virus (HCV) is an important antiviral target. Currently, three PIs have been approved for therapy of chronic hepatitis C, and several others are in development. NS3-dependent cleavage of the HCV polyprotein is required to generate the mature nonstructural proteins that form the viral replicase. Inhibition of protease activity can block RNA replication by preventing expression of mature replicase components. Like many viral proteins, NS3 is multifunctional, but how PIs affect stages of the HCV life cycle beyond polyprotein processing has not been well studied. Using cell-based assays, we show here that PIs can directly inhibit viral RNA synthesis and also block a late stage in virus assembly/maturation at clinically relevant concentrations.

Measuring patient satisfaction in orthopaedic surgery.

In addition to their wish to understand the clinical results of orthopaedic interventions, clinicians, patients, and payers are increasingly interested in patient satisfaction, both with the process of care and with outcomes. The construct of satisfaction is complex and depends on the context in which care takes place, including the nature of treatment, its setting, and most importantly the expectation of patients prior to treatment. The characteristics of scales that are effective measures of satisfaction are the same as those of all effective measurement instruments--i.e., reliability, validity, and responsiveness. Measurement of patient satisfaction may be especially important in evaluations of established procedures and processes so that the value of those procedures and processes to patients can be more completely understood.

Clinical practice guidelines: what are they and how should they be disseminated?

Clinical practice guidelines summarize the available evidence for patient management in a format that is easy for clinicians to use. These guidelines usually use methodologically rigorous principles for retrieving and evaluating the literature and for establishing consensus among work group members, but implementation by clinicians is often incomplete. The reasons why guidelines fail to gain widespread acceptance vary with the topic and clinician group. Successful dissemination of practice guidelines requires an understanding of the barriers to implementation and the use of multiple strategies to address these. This article examines the factors affecting implementation and the approaches to overcoming these obstacles.

Continuous infraclavicular brachial plexus blockade: effect on survival of replanted digits.

In replantation surgery, the use of continuous brachial plexus blockade (CBPB) is popular as it improves postoperative analgesia and vascular flow. The aim of our study was to determine whether CBPB may affect the odds of survivability of replanted digit(s). A four-year retrospective chart review was performed and various parameters affecting replant survival were examined. Outcome was recorded as successful if the transplanted digit(s) survived six months after discharge. All the independent variables were forced into a regression model without using a specific variable selection algorithm. The data for 146 patients was obtained from our chart review. The success rate of replanted digits in the patients reviewed was 65.8%. The logistic regression model showed a relation between the number of digits injured and replanted digit(s) survival. Our study showed that CBPB has no effect on the survivability of the replanted digit(s) till six months after hospital discharge.